Fungicidal compositions and methods utilizing 25-azasterol and HMG-CoA synthase inhibitors

ABSTRACT

Novel fungicidal compositions comprising a 25-azasterol compound and a HMG-CoA synthase inhibitor compound are disclosed.

BACKGROUND OF THE INVENTION

Systemic fungal infections caused by Candida species, Cryptococcusneoformans, Histoplasma capsulatum and the like are often serious orfatal. There continues to be a need for an antifungal agent which iseffective and at the same time non-toxic to the patient being treated. Anumber of known antifungal agents while effective in eliminating thedisease are of limited usefulness because of toxic or other undesirableside reactions. In many cases the toxicity is related to the amount ofthe drug and could be eliminated or reduced if a less amount of the drugcould be employed. Thus, it would be desirable if a combination could befound which is synergistic or which has a second component that has apotentiating effect on the known antifungal agent thereby reducing theamount of drug required with concomitant reduction or elimination ofside reactions.

There are reports in the literature that by administering amphotericinwith other antibiotics, Candida albicans is sensitized to antifungalagents. Thus, for example, minocycline and amphotericin B have beenfound to be a synergistic composition against Candida albicans, Leev etal, Brit. J. Infect. Dis., 136, 263-270 (1977). However, combinationswith amphotericin is not satisfactory because of the toxicity ofamphotericin to human beings.

STATEMENT OF THE INVENTION

The present invention concerns improved compositions and methods usefulfor the control of fungi and particularly for the treatment of mycoticinfections made possible by the discovery that when certain azasterolcompounds are employed in combination with certain fungistaticcompounds, there is potentiation in the activity of these compoundsresulting in superior antifungal combinations and in some casesresulting in useful fungicidal combinations. Some azasterols may showsome antifungal properties in which case the potentiated compositionsmay be considered a synergistic composition.

DESCRIPTION OF THE INVENTION

According to the present invention it has been discovered that theantifungal properties of an antifungal agent may be potentiatede bycoadministering a 25-azasterol compound with the antifungal agent. Thus,when a 25-azasterol compound, at a concentration not inhibitory to thegrowth of fungi is employed with a subfungistatic amount of anantifungal agent, the sensitivity of fungal organisms to the antifungalagents is unexpectedly significantly increased providing a synergisticantifungal composition. It has further been discovered that with certainfungistats the combinations have resulted unexpectedly in fungicidalcompositions.

In view of the effectiveness of the combination against fungi causingmycotic infections, the present invention is further directed to methodsand compositions for combatting fungi causing mycotic infectionscomprising treating the infected site with an antifungally effectiveamount of a composition comprising a subfungistatic amount of anantifungal compound and a 25-azasterol compound in an amount notinhibitory to fungal growth. The invention is further directed to amethod for treating mycotic infections comprising administering to asubject in need of such treatment, an antifungally effective amount of acomposition comprising a subfungistatic amount of an antifungal compoundand an amount of 25-azasterol not inhibitory to fungal growth.

Fungistats which have been potentiated most effectively and indeedunexpectedly to give rise to fungicidal compositions are thosefungistats which act on the enzymes in the biochemical pathway prior tothe formation of lanosterol. Inhibitors of enzymes in the mevalonic acidsynthesis such as HMG-CoA synthase, β-kethothiolase (β-ketoacyl-coenzymeA thiolase) and HMG-CoA reductase as well as those which inhibit enzymeswhich act at later stages in the pathway to lanosterol includingsqualane epoxidase are particularly imp/rtant in the formation of novelfungicidal compositions. Since all of these enzymes are enzymes in thepathway of lanosterol synthesis, the antifungal agents which act onthese enzymes when spoken of in general terms will be referred to aslanosterol synthesis inhibitors. In view of the effectiveness of25-azasterols on potentiating the fungistats which are lanosterolsynthesis inhibitors, the invention is further directed to methods andcompositions for treating mycotic infections comprising a subfungistaticamount of a lanosterol synthesis inhibitor and a 25-azasterol compoundin an amount non-inhibitory to fungal growth.

Although mycotic infections may be alleviated by fungistaticcompositions, there is no cure except with fungicidal compositions. Avery special aspect of the present invention are new fungicidalcompositions comprising a 25-azasterol compound and a HMG-CoA synthaseinhibitor, or a β-ketothoilase inhibitor, or a HMG-CoA reductaseinhibitor or a squalene epoxidase inhibitor.

The "25-azastrerol compound"useful in the improved antifungalcompositions and methods of the present invention are characterized by ahydroxyl group or an esterified hydroxyl group in the 3-position, anunsaturation in Ring B and a nitrogen at the 25 position in the sidechain of the steroid ring system as may be seen from the followingformula (I): ##STR1## wherein R is hydrogen or lower acyl, X is CH, CH₂or O; and the on the side chain indicates that the bond may be a singleor a double bond provided that when X is O, or CH₂, it is a single bond.

Except as hereinafter indicated, the azasterol compounds are compoundsavailable or reported in the literature or are ester derivatives ofknown compounds readily prepared by conventional esterification methods.The following are representative examples of specific 25-azasterolswhich are particularly useful in the present invention;

(a) 25-azacholesterol;17β-[[3-(dimethylamino)-propyl]methylamino]androst-5-en-3β-ol ##STR2##

(b) N,N-dimethyl-3β-hydroxy-5,22(Z)choladiene-24-amine;(3β22Z)-24-(dimethylamino)chola-5,22-diene-3-ol ##STR3## The foregoingmay be prepared by the Wittig reaction on an i-steroid carboxaldehyde,followed by rearrangement and hydrolysis as described in copendingapplication U.S. Ser. No. 169,699 in the name of N. G. Steinberg, nowabandoned.

(c) N,N-diemthyl-3β-acetoxy-5,22(Z)choladiene-24-amine;(3β,22Z)-24-(dimethylamino)chola-5,22-dien-3-ol acetate. ##STR4## Thepreparation of the acetate is also the subject of U.S. Ser. No. 169,699in the name of N. G. Steinberg, now abandoned.

(d) 25-aza-22-oxacholesterol;(3,20)-20-[2-(dimethylamino)ethoxy]pregn-5-en-3-ol ##STR5## Thepreferred 25-azasterol compound is 25-azacholesterol (Compound Ia).

The antifungal compounds which may be potentiated by the 25-azasterolcompounds are diverse in structure but are useful especially againstorganisms causing mycotic infections. A number of the suitableantifungal compounds are those known to be inhibitors of enzymes in thebiochemical pathway to lanosterol. However, a number of antifunggalagents which are not inhibitors of enzymes in the biochemical pathway tolanosterol or where mode of antifungal action is not established butwhich are known or can be shown to possess antifungal properties havealso been found to be potentiated and to form useful synergisticantifungal compositions. These compounds of diverse structure which arethe main antifungal component in the methods and compositions of thepresent invention are non-steroidal in nature, although as previouslynoted, some of since the 25-azasterol compounds have some antifungalproperties. Representative of the compounds which are potentiated by the25-azasterol compound include a number of new fungistats as well asestablished fungistats recognizable by USAN or generic names. Thefollowing representative compounds illustrate the diverse nature of theantifungal agents which may be potentiated but are not to be construedas being limited thereto.

(1) 11-(3-hydroxymethyl-4-oxo-2-oxetanyl)-7-methyl-2,4-undecadienoicacid. (Compound A) ##STR6## This compound is reported in J. Chem. Soc.(c), 1971, 3888; the antifungal properties is the subject of U.S. Ser.No. 825,496, filed Feb. 3, 1986, now abandoned and the HMG-CoA synthaseinhibitor property, the subject of U.S. Ser. No. 21,848, filed Mar. 4,1987 now U.S. No. 4,847,271, Jul. 11, 1989.

(2) 5-(1-hydroxy-2,4,6-heptatriynyl)-2-oxo-1,3-dioxolane-4-heptanoicacid. (Compound B) ##STR7## The compound may be prepared by thecultivation of microorganism ATCC 53,614, 53,615 or 53,616 followed byisolation as described together with antifungal properties in U.S. Ser.No. 53,920, filed May 26, 1987 now U.S. No. 4,806,565, Feb. 21, 1989.The β-ketothiolase activity is subject of U.S. Ser. No. 53,973, filedMay 26, 1987 now U.S. No. 4,780,311.

(3) 1-methyl-2-nonyl-5-(phenylmethyl)-3-pyrrolidinol (Compound C)##STR8## The compound may be prepared by the cultivation ofmicroorganism ATCC 22947 followed by isolation as described togetherwith antifungal properties in copending application U.S. Ser. No.172,164, filed in the name of R.E. Schwartz et al now U.S. Pat. No.4,847,284, Jul. 11, 1989.

(4)(E)-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphthalenemethylamine;terbinafine (Compound D) ##STR9##

(5)1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl-2-methylbutanoate; lovastatin (Compound E) ##STR10##

(6) 1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylicacid; nalidixic acid (Compound F) ##STR11##

(7)5-chloro-6-(7,8-epoxy-10-hydroxy-2-oxo-3,5-undecadienyl)-β-resorcyclicacid μ-lactone; monorden (Compound G) ##STR12##

(8) 2-(4-thiazolyl)-1H-benzimidazole; thiabendazole (Compound H)##STR13##

(9)4-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]2,6-piperidinedione;cycloheximide (Compound J) ##STR14##

(10)4,7-bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-actahydro-3,10,12,12a-tetrahydroxy-1-dioxo-2naphthacenecarboxamide;minocycline (Compound K) ##STR15##

(11)4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide;tetracycline (Compound L) ##STR16##

(12) N-methyl-N-(3-phenyl-2-propenyl)-1-naphthalenemethanamine;naftifine (Compound M) ##STR17##

(13) 2-(p-methoxybenzyl)-3,4-pyrrolidinediol-3-acetate; anisomycin(Compound N) ##STR18##

The foregoing compounds hereinafter may be identified by the generic ofUSAN name or by "Compound" followed by the letter designations.

As previously indicated, the combinations which are noteworthy are thosecombinations of 25-azasterol compounds with inhibitors of enzymes in thebiochemical pathway in lanosterol synthesis. These combinations showfungicidal properties as well as synergistic antifungal properties.Especially noteworthy are combinations of 25-azasterol compounds withinhibitors of mevalonic acid synthesis, i.e., a 25-azasterol compoundwith a HMG-CoA synthase inhibitor compound; a 25 -azasterol compoundwith a β-ketothiolase inhibitor compound and a 25-azasterol compoundwith a HMG-CoA reductase inhibitor compound. Particularly usefulspecific combinations are 25-azacholesterol as the 25-azasterol compoundwith 11-(3-(hydroxymethyl)-4-oxo-2-oxetanyl)-7-methyl-2,4-undecadienoicacid as (Compound A) the HMG-CoA synthase inhibitor compound, with5-(1-hydroxy-2,4,6-heptatriynyl)-2-oxo-1,3-dioxalane-4-heptanoic acid(Compound B) as the β-ketothiolase inhibitor compound, and with1,2,3,7,8,8a-hexahydro-3,7-dimethyl8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl-2butanoate(Compound E; lovastatin) as the HMG-CoA reductase inhibitor compound.Also noteworthy are combinations of 25-azasterol compounds with squaleneepoxidase inhibitor compounds such as(E)-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphthalene-methylamine(Compound D; terbinafine) andN-methyl-N-(3phenyl-2-propenyl)-1-naphthalenemethanamine hydrochloride(Compound M; naftifine). Especially useful of the latter is thecombination of 25-azacholesterol and terbinafine.

The synergistic antifungal and fungicidal combinations of the presentinvention are effective in the treatment of mycotic infections caused bysuch fungal organisms as those of the Candida species, for example, C.albicans, C. parapsilosis, C. tropicalis, C. pseudotropicalis, C.krusei, C. rugosa, C. guilliermondii, C. stellatoidea; those of theAspergillus species such as A. fumigatus; and other disease-causingfungi such as Cryptococcus neoformans; Torulopsis glabrata; Rhizopusrhizopodiformis; Coccidioides immitis; Sporothrix schenkii; Histoplasmacapsulatum; and Blastomyces dermatitidis.

The method for potentiating the antifungal effectiveness of anantifungal compound comprising employing a subfungistatic amount of anantifungal compound together with a 25-azasterol compound which if ithas some fungistatic property is employed at a concentration notinhibitory to the growth of fungi. The potentiated combination may thenbe employed to control fungal growth by administering to or directing toa site where control of fungi is desired, an antifungally effectiveamount of a composition comprising a 25-azasterol compound in an amountnon-inhibitory to fungal growth and an antifungal compound.Alternatively, each compound may be administered sequentially. Themethod is particularly directed to treating subjects with mycoticinfections to control fungal growth and the disease caused by fungi bycomprising administering to said subjects an antifungally effectiveamount of a composition comprising a 25-azasterol compound together withan antifungal agent. The application may be made at a site remote fromthat of the infection such as would be the case with oral or parenteraladministration, or directly at the site infected with fungi. The agentsmay be administered with or without, preferably with, a pharmaceuticallyacceptable carrier in the amounts hereinafter set forth. By theadministration of the amounts of the agents as hereinafter set forth, apotentiated or synergistic fungistatic interaction of the drugs isachieved which is wholly unexpected.

With some combinations, namely, azasterols with lanosterol synthesisinhibitors, a fungicidal effect also may be achieved, usually at higherconcentrations. With combinations which have a fungicidal effect, theinvention is directed to killing fungi by administering to the siteinfected with fungi, the fungicidal combination. It is further directedto treating subjects with mycotic infection to eradicate, thedisease-causing fungi by administering a fungicidal compositioncomprising an azasterol compound and a HMG-CoA synthase inhibitor, anazasterol compound and a β-ketothiolase inhibitor an azasterol compoundand a HMG-CoA reductase inhibitor or an azasterol compound and asqualene epoxidase inhibitor. As with fungistatic compositions, theapplication may be made at a site remote from the infection as in oralor parentaral administration, or may be made directly at the site ofinfection. The administration may be made with or without a carrier andin amounts as hereinafter detailed. By these operations, a whollyunexpected eradication of fungi has been achieved.

The effectiveness of the combination generally depends on the originalsusceptibility of the particular organism or strain of organism to thefungistat to be employed in combination with the azasterol compound.Thus, although the azasterol compound has a potentiating effect onfungistats generally, the apparent greater effect of particularcombinations may depend on the original susceptibility of the organismto the unmodified fungistat. In the case of fungistats which are knownto be lanosterol synthesis inhibitors, it has been found with thesefungistats that not only a synergistic fungistatic combination isobtained but also a fungicidal combination is obtained.

The potentiation of the antifungal properties of an antifungal agent bycoadministering a 25-azasterol compound and the production of asynergistic antifungal as well as in many instances of a fungicidaleffect may be illustrated by the in vitro interaction studies for thedetermination of activity. In these tests against representative fungalorganisms known to be the causative agent of mycotic infections,including Candida albicans, other Candida speices, and a number of otherfungi, synergistic antifungal properties have been demonstrated with a25-azasterol compound together with various fungistatic lanosterolsynthesis inhibitor compounds.

Representative antifungal properties of the combination of a25-azasterol compound and an antifungal agent may be seen in thefollowing examples, which are deemed to be representative but which arenot to be construed as limiting.

EXAMPLE I Potentiation of Anti-Candida Activity by 25-Azacholesterols

Using standard a disk diffusion assay in which the agar was supplementedwith 25-azacholesterol, and using two strains of Candida albicans, MY992 and MY 1055, drugs were tested to determine the extent ofpotentiating effect of the 25-azacholesterol on the antifungalproperties of the drugs. Representative potentiating effect of25-azacholesterol on the anti-Candida activity of some antifungal agentsmay be seen from the following table:

                  TABLE I                                                         ______________________________________                                                      Zone diameters (mm)                                                           MY 992    MY 1055                                                                  25-Azacholesterol                                          Antifungal      μg/ml        μg/ml                                      Drug      μg/disk                                                                              0     20*  Δ                                                                            0    20*  Δ                         ______________________________________                                        Nalidixic acid                                                                          50        0     25   25   15   25   10                                        10        0     16   16   0    15   15                              Lovastatin                                                                              50        17    25   8    17   25   8                                         10        12    18   6    12   20   8                               Terbinafine                                                                             50        19    30   14   17   31   14                                        10        15    24   11   15   27   12                              Compound C                                                                              5         0     34   31   0    40   40                                        0.5       0     30   30   0    35   35                              Nikkomycin                                                                              50        0     18   18   0    16   16                                        10        0     0    0    0    0    0                               Monorden  50        15    22   7    15   18   3                                         10        0     20   20   0    17   17                              Thiabendazole                                                                           50        0     20   20   0    20   20                                        10        0     17   17   0    18   18                              Cycloheximide                                                                           50        0     17   17   0    15   15*                             Minocycline                                                                             50        14    25   11   15   20   -5                              Tetracycline                                                                            50        0     13   13   0    0    0                               ______________________________________                                         *At 20 μg/ml, 25azacholesterol alone did not inhibit the growth of C.      albicans MY 992 or MY 1055.                                              

EXAMPLE II

Sabouraud dextrose agar was inoculated with Candida albicans MY 992 to1×10⁴ colony forming units/milliliter (cfu/ml). The azasterols,25-azacholesterol (Compound Ia) and 25-aza-22-oxacholesterol (CompoundId), dissolved in ethanol:Brij 58 (1:1, v/v), were added in a volumeequivalent to 10 μl/ml. Assay plates were poured. Sensitivity disksprepared to provide stated concentrations of the drug were placed on theplates. The plates were incubated for 17 hours at 37° C. At the end ofthis period, zones of inhibition were measured. The effect of theazasterols in increasing the sensitivity of the organism to the drug maybe seen in Table II.

                  TABLE II                                                        ______________________________________                                                     Zone of Inhibition                                                            against                                                                       C. albicans MY 992                                                                           Compound                                                                              Compound                                  Drug      μg/disk                                                                             Control  Ia      Id                                        ______________________________________                                        Anisomycin                                                                              50       0        15      14                                        Cycloheximide                                                                           50       0        16      14                                        Nalidixic acid                                                                          50       0        25      20                                        Nikkomycin                                                                              50       0        30      31                                        Tetracycline                                                                            50       0        16      15                                        Lovastatin                                                                              50       13       21      18                                        "         5        0        15      15                                        Naftifine 50       0        20      20                                        "         5        0        12      12                                        Terbinafine                                                                             50       17       25      25                                        "         5        12       19      20                                        ______________________________________                                    

EXAMPLE III

A disk diffusion assay was carried out in which several azasterolcompounds were employed with several antifungal agents to determine thepotentiating properties of the azasterol compounds.

The azasterols were titrated in Sabouraud dextrose agar which wasinoculated with C. albicans MY992. Sensitivity disks containingantifungal agent at 50 μg/disk were applied to the plates. The plateswere incubated at 37° C. for 18 hours. At the end of this period theplates were read for zones of inhibition.

The results of azasterol compounds: 25-azacholesterol (Compound Ia),N,N-dimethyl-3β-acetoxy-5,22(Z)-choladiene-24-amine (Compound Ic) andN,N-dimethyl-3β-hydroxy-5,22(Z)-choladiene-24-amine (Compound Ib) withantifungal agents: cycloheximide; nalidixic acid and terbinafine areseen in Table III. (Compound Ic inhibited growth at 25 μg/ml whenemployed alone.)

                  TABLE III                                                       ______________________________________                                                         Cyclo-    Nalidixic                                          Azasterol        heximide  acid    Terbinafine                                Compound μg/ml                                                                              50 μg/disk                                                                           50 μg/disk                                                                         50 μg/disk                              ______________________________________                                        Control          0         0       17                                         Compound Ia                                                                            25      16 h      27      26                                                  6.25    14 h      25      24                                                  1.56    13 h      22      25                                                  0.39    0         21      24                                         Compound Ic                                                                            25      Inhibited                                                                     growth                                                                6.25    14 h      19      16                                                  1.56    0         15      18                                                  0.39    0         11      18                                         Compound Ib                                                                            25      15 h      36      29                                                  6.25    15 h      35      26                                                  1.56    0         23      21                                                  0.39    0         18      22                                         ______________________________________                                    

EXAMPLE IV A. Synergistic Effect

Minimum inhibitory concentration (MIC) of nalidixic acid, nikkomycin,lovastatin and terbinafine alone and in combination with25-azacholesterol were determined.

Synthetic Medium (SM, Difco yeast nitrogen base supplemented with 0.5percent dextrose) and Complex Medium (CM, Difco Sabouraud dextrose agar)were inoculated with 1×10⁴ cfu/ml (colony forming units per milliliter)of exponential phase Candida albicans MY 992. Dilution tubes containing10 μl/ml of dimethyl sulfoxide (DMSO) and 10 μl/ml of ethanol-Brij 58(1:1, v/v) were prepared. The test drugs were titrated from 400 to 1.56μg/ml. The tubes were incubated for 17 hours at 37° C., read and theminimum inhibitory concentrations determined. The MIC was taken as theminimum concentration which prevented visible growth. The results areseen in Table IVA.

                  TABLE IVA                                                       ______________________________________                                                     Minimum Inhibitory Concentration                                              (MIC) of Drug                                                                 μg/ml                                                                      Concn of 25-Azacholesterol (μg/ml)                            Drug      Medium   0          1.25   12.5                                     ______________________________________                                        Nalidixic Acid                                                                          SM       200        100    100                                                CM       >400       100    100                                      Nikkomycin                                                                              SM       >400       50     50                                                 CM       >400       200    200                                      Lovastatin                                                                              SM       400        100    50                                                 CM       200        100    50                                       Terbinafine                                                                             SM       200        50     25                                                 CM       200        25     25                                       ______________________________________                                    

B. Fungicidal Effect

The fungicidal effect of the drugs alone and in combination with25-azacholesterol were determined by diluting the titration tubes whichhad been used to determine the minimum inhibitory concentrations.

The test samples were diluted by 10-fold increments in microtiterdishes. A 5 microliter aliquot of each dilution was stamped on Sabourauddextrose agar and the plates incubated for 24 hours at 37° C. Thepresence of viable cells in the MIC tubes was noted and the minimumfungicidal concentration (MFC) determined. The results are seen in TableIVB.

                  TABLE IVB                                                       ______________________________________                                                   Minimum Fungicidal Concentration                                              (MFC) of Drug                                                                 μg/ml                                                                      Concn of 25-Azacholesterol (μg/ml)                              Drug         0           12.5                                                 ______________________________________                                        Nalidixic Acid                                                                             >400        >400                                                 Nikkomycin   >400        >400                                                 Lovastatin   >400         100                                                 Terbinafine   400          25                                                 ______________________________________                                    

EXAMPLE V

The potentiating effect of 25-azacholesterol on the minimal inhibitoryconcentration (MIC) or minimal fungicidal concentration (MFC) of certainantifungal agents against C. albicans MY 1055 are seen in the followingexperiments.

C. albicans MY 1055 was grown overnight in Difco yeast nitrogen basesupplemented with 0.5 percent glucose at 37° C. The culture was dilutedinto fresh medium to 1×10⁴ cfu/ml. A one-milliliter aliquot was added totubes containing drugs serially diluted by four-fold increments. The25-azacholesterol was used as the dihydrochloride salt. Afterinoculation, the tubes were incubated for 24 hours at 37° C. at 300 rpm.The MIC was determined as the lowest concentration preventing visiblegrowth.

The minimum fungal concentration (MFC) was determined by diluting thedrug-treated overnight cultures by 10-fold increments in microtiterdishes. Five microliters of each dilution was stamped on Sabourauddextrose agar. The plates were incubated for 24 hours at 37° C. andobserved for growth. The MFC was determined as the lowest concentrationto kill which was greater than or equal to 2 logs of the initial colonyforming units.

The results are seen in Table V.

                  TABLE V                                                         ______________________________________                                                 Minimum                                                                       Inhibitory      Fungicidal                                                    Concn           Concn                                                         μg/ml        μg/ml                                                      25-Azacholesterol                                                             μg/ml                                                             Drug       0        25       0      25                                        ______________________________________                                        Nalidixic Acid                                                                           400      100      400    400                                       Lovastatin  25      1.56     100     25                                       Terbinafine                                                                              100      1.56     400     25                                       Compound C 400       25      >400   >100                                      Nikkomycin 100      100      400    >100                                      Monorden    25      6.25     100    100                                       Minocycline                                                                              >400     400      >400   400                                       ______________________________________                                    

EXAMPLE VI

In operations carried out in the manner described in Example V, minimuminhibitory concentration and minimum fungicidal concentrations of thecombinations of 25-azacholesterol with5-(1-hydroxy-2,4,6-heptatriynyl)-2-oxo-1,3-dioxalane-4-heptanoic acidcarbonate salt (Compound B) and with11-(3-(hydroxymethyl)-4-oxo-2-oxetanyl)-7-methyl-2,4-undecadienoic acid(Compound A) against Candida albicans MY1055 were determined. Theresults are seen in Table VI.

                  TABLE VI                                                        ______________________________________                                                   MIC          MFC                                                              μg/ml     μg/ml                                                         25-Azacholesterol (μg/ml)                                       Drug         0       25         0    25                                       ______________________________________                                        Compound B   1.25    <0.31       5.0 <0.31                                    Compound A   3.1     <3.1       12.5 <3.1                                     ______________________________________                                    

EXAMPLE VII

The enhanced activity of combinations against a wide spectrum of yeastsand filamentous fungi may be seen in the combinations of25-azacholesterol with terbinafine, monorden, and Compound C in an agardilution assay. The method employed was as follows: 25-azacholesterolwas solubilized in distilled water and concentrations in the range offrom 1.28 to 0.00063 mg/ml were first prepared and then diluted in agarmedium to obtain test concentrations of 128 to 0.063 mg/ml. Terbinafine,monorden and Compound C were solubilized in 10 percent dimethylsulfoxide and thereafter diluted as with the 25-azacholesterol exceptthat the maximum final concentration of Compound C was 64 μg/ml.

Each diluted drug was added to cooled, molten yeast nitrogen base plusglucose agar (1.0 ml of drug plus 9.0 ml agar). Appropriate solvent andmedia controls (drug free) also were prepared. Prepared plates werestored in the dark at room temperature overnight prior to use.

The yeast cultures, maintained in yeast maltose (YM) broth, weretransferred to fresh YM medium and incubated overnight at 37° C. withshaking (250 rpm). After incubation, each culture was diluted in sterilesaline to yield final concentrations of 3×10⁵ to 3×10⁶ colony formingunits per milliliter (cfu/ml). The isolates of two species ofAspergillus and of Penicillium were maintained on potato dextrose agarslants and spore suspensions made following vigorous shaking withsterile glass beads. The spore preparations were used as the inocula forthese three filamentous fungi.

Each prepared plate was inoculated with 21 yeast-like and filamentousfungi using a Denley Multipoint Inoculator (Denley, Sussex, England).The inoculator delivers approximately 0.001 ml to the agar surfaceresulting in inoculation of from 3×10² to 3×10³ colony forming units.The plates were incubated at 28° C. for 48 hours. The minimum inhibitoryconcentrations (MICs) were recorded as the lowest concentration of drugshowing no growth or less than three cfus/spot.

Potentiation of activity by the azasterol compound was seen in allcombinations against some of the organisms. Combinations with monorden,and terbinafine gave greatly increased activity against almost everyorganism tested.

The results with monorden, Compound C and terbinafine are seen in TablesVIIA, VIIB, and VIIC. In all combinations, tests were carried out inwhich 25-azacholesterol (AC) was maintained at 128 μg/ml. Monorden (M)or terbinafine (T) was titrated from 128 μg/ml to 0.063 μg/ml, whichCompound C (Cpd C) was titrated from 64 μg/ml to 0.063 μg/ml. Withterbinafine additional tests were carried out in which 25-azacholesterolwas maintained at 32 μg/ml or 8 μg/ml while terbinafine was titratedfrom 128 μg/ml to 0.063 μg/ml. The minimum inhibitory concentration inthe combination column is that of the titrated fungistat in the mixture.In combinations with terbinafine no significant reduction inpotentiating activity was observed by reducing 25-azacholesterolconcentrations from 128 μg/ml to 8 μg/ml.

A similar test employing 25-azacholesterol with nalidixic acid showedsimilar but less potent potentiating effect. The pattern of activity wassimilar to that of the combination with monorden exhibiting highpotentiation against C. pseudotropicalis, C. krusei and Penicilliumitalicum. However, unlike in the combination with monorden,25-azacholesterol showed little potentiating effect on nalidixic acidagainst most strains of Candida albicans.

                  TABLE VIIA                                                      ______________________________________                                                         MINIMUM                                                                       INHIBITORY                                                                    CONCENTRATION                                                                 (μg/ml)                                                                              M                                                                     AC      (with    M                                         FUNGUS             (alone) 128AC)   (alone)                                   ______________________________________                                        Cryptococcus neoformans                                                                     MY1051   >128    <0.063 16                                      Cr. neoformans                                                                              MY1146   >128    0.5    16                                      Candida albicans                                                                            MY1058   >128    1      32                                      C. albicans   MY1055   >128    2      32                                      C. albicans   MY0992   >128    4      64                                      C. albicans   MY1013   >128    2      32                                      C. albicans   MY1029   >128    2      32                                      C. parapsilosis                                                                             MY1009   >128    2      64                                      C. parapsilosis                                                                             MY1010   >128    0.25   32                                      C. tropicalis MY1011   >128    8      >128                                    C. tropicalis MY1012   >128    0.25   1                                       C. pseudotropicalis                                                                         MY1040   <0.063  <0.063 >128                                    C. krusei     MY1020   <0.063  <0.063 64                                      C. rugosa     MY1022   >128    32     >128                                    C. quilliermondii                                                                           MY1019   >128    4      >128                                    C. stellatoidea                                                                             MY1018   >128    8      >128                                    Torulopsis glabrata                                                                         MY1059   >128    16     >128                                    Sac. cerevisiae                                                                             MY1027   >128    0.125  32                                      Aspergillus fumigatus                                                                       MF4839   >128    4      32                                      A. flavus     MF0383   >128    4      32                                      Penicillium italicum                                                                        MF2819   1       <0.063 16                                      ______________________________________                                    

                  TABLE VIIB                                                      ______________________________________                                                          MINIMUM                                                                       INHIBITORY                                                                    CONCENTRATION                                                                 (μg/ml)                                                                              Cpd C                                                                 AC      (with    Cpd C                                    FUNGUS              (alone) 128AC)   (alone)                                  ______________________________________                                        Cryptococcus neoformans                                                                      MY1051   >128    4      >64                                    Cr. neoformans MY1146   >128    64     >64                                    Candida albicans                                                                             MY1058   >128    2      >64                                    C. albicans    MY1055   >128    <0.063 >64                                    C. albicans    MY0992   >128    >64    >64                                    C. albicans    MY1013   >128    1      >64                                    C. albicans    MY1029   >128    <0.063 >64                                    C. parapsilosis                                                                              MY1009   >128    0.125  >64                                    C. parapsilosis                                                                              MY1010   >128    <0.063 >64                                    C. tropicalis  MY1011   >128    16     >64                                    C. tropicalis  MY1012   >128    32     >64                                    C. pseudotropicalis                                                                          MY1040   <0.063  <0.063  32                                    C. krusei      MY1020   <0.063  <0.063 >64                                    C. rugosa      MY1022   >128    <0.063 >64                                    C. quilliermondii                                                                            MY1019   >128    <0.063 >64                                    C. stellatoidea                                                                              MY1018   >128    16     >64                                    Torulopsis glabrata                                                                          MY1059   >128    >64    >64                                    Sac. cerevisiae                                                                              MY1027   >128    <0.0630                                                                               32                                    Aspergillus fumigatus                                                                        MF4839   >128    >64    >64                                    A. flavus      MF0383   >128    >64    >32                                    Penicillium italicum                                                                         MF2819   1       <0.063 >64                                    ______________________________________                                    

                                      TABLE VIIC                                  __________________________________________________________________________                MINIMUM INHIBITORY CONCENTRATION (μg/ml)                                        AC     T      T      T     T                                 FUNGUS      (alone)                                                                            (with 128AC)                                                                         (with 128AC)                                                                         (with 32AC)                                                                          (with 8AC)                                                                          (alone)                           __________________________________________________________________________    Cryptococcus neoformans                                                                   MY1051                                                                             >128   1      1      2     8                                 Cr. neoformans                                                                            MY1146                                                                             >128   2      2      2     32                                Candida albicans                                                                          MY1058                                                                             >128   4      4      4     128                               C. albicans MY1055                                                                             >128   4      4      4     128                               C. albicans MY0992                                                                             >128   >128   >128   >128  >128                              C. albicans MY1013                                                                             >128   4      4      4     64                                C. albicans MY1029                                                                             >128   2      2      2     128                               C. parapsilosis                                                                           MY1009                                                                             >128   0.125  <0.063 0.125 4                                 C. parapsilosis                                                                           MY1010                                                                             >128   0.125  <0.063 0.25  4                                 C. tropicalis                                                                             MY1011                                                                             >128   16     16     16    128                               C. tropicalis                                                                             MY1012                                                                             >128   4      4      4     8                                 C. krusei   MY1020                                                                             0.5    <0.063 <0.063 <0.063                                                                              64                                C. rugosa   MY1022                                                                             >128   4      4      4     >128                              C. quilliermondii                                                                         MY1019                                                                             >128   1      0.5    1     32                                C. stellatoidea                                                                           MY1018                                                                             >128   8      8      8     64                                Torulopsis glabrata                                                                       MY1059                                                                             >128   >128   >128   >128  >128                              Sac. cerevisiae                                                                           MY1027                                                                             >128   <0.063 16     32    128                               Aspergillus fumigatus                                                                     MF4839                                                                             >128   0.125  0.125  <0.063                                                                              0.5                               A. flavus   MF0383                                                                             >128   <0.063 <0.063 <0.063                                                                              0.5                               Penicillium italicum                                                                      MF2819                                                                             >128   <0.063 <0.063 <0.063                                                                              16                                __________________________________________________________________________     .sup.a In the combination assay, the 25azacholesterol concentration was       assayed at either 128 AC(128), at 32 AC(32) or at 8 AC(8) mg/ml; and          terbinafine T was titrated from 128 μg/ml to 0.063 μg/ml.          

From the test results, the known dosage ranges of the variousfungistatic compounds, and the concentration of the 25-azasterolcompound which is not inhibitory to the growth of fungi, a synergisticantifungal or fungicidal composition may be achieved for controllingmycotic infections, the fungistatic or fungicidal effect being dependentprimarily on the antifungal agent. Potentiation may be obtained when theazasterol compound is employed in an amount of from about 1/4 to 1/2 oreven 1/30th of the amount of the non-steroidal antifungal agent. For thecombinations to produce a fungicidal effect, the amount of the25-azasterol compound may be increased over that needed to produce thesynergistic antifungal effect. However, it is to be kept in mind thatsince the antifungal property resides primarily in the non-steroidalentity and it is oftentimes the desire to decrease the amount of thisnon-steroidal entity to take advantage of its antifungal property whilereducing undesired side reactions, the actual amount of the steroidalagent is not necessarily of primary consideration and the desired usefulpotentiating effect may be obtained with an amount of the steroidalentity in weight excess rather than in a small fraction by weight. Theactual ratio of the steriodal potentiating agent to the antifungal agentwill be dependent not only on the non-steroidal fungistat but on theparticular organism being controlled. Thus, the actual ratio is notcritical. The present invention contemplates use of a subfungistaticamount of the fungistat selected together with an amount of azasterolcompound which is non-inhibitory to fungal growth. For therapeuticcontrol of mycotic infections from about 1.5 to about 5.0 mg/kg of bodyweight of the non-steroidal antifungal agent and similarly an amount of1.5 to 5.0 mg/kg of body weight of the azasterol compound may beadministered per day while considering patient's health, weight, age andother factors which influence response to a drug as well as theparticular drug to be employed. The amounts when expressed as dosessuitable for use in human subjects are in the range of from about 100 toabout 400 mg of the non-steroidal antifungal agent and 50 to 400 mg ofthe azasterol compound given BID by oral or parenteral route.

The outstanding properties are most effectively utilized when theazasterol compound and a non-steroidal antifungal agent are formulatedinto novel pharmaceutical compositions with a pharmaceuticallyacceptable carrier according to conventional pharmaceutical compoundingtechniques.

The synergistic combination may be formulated for injection and may bepresented in unit dosage form in ampoules or in multidose containers, ifnecessary with an added preservative. The compositions may also takesuch forms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulating agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredients may be in powder form for reconstituting with a suitablevehicle prior to parenteral or oral administration.

For parenteral applications the drugs are preferably formulated inconventional parenteral solutions such as 0.85 percent sodium chlorideor 5 percent dextrose in water, with formulation aids such asdesoxycholate or other pharmaceutically acceptable compositions.

The compounds also may be prepared in tablet or capsule form as well asin liquid form for oral administration. In preparing the compositions inoral dosage form, the component drugs are intimately admixed with any ofthe usual pharmaceutical media, including for liquid preparations,liquid carriers such as water, glycols, oils, alcohols, and the like,and for solid preparations such as capsules and tablets, solid carrierssuch as starches, sugars, kaolin, ethyl cellulose, generally withlubricant such as calcium stearate, together with binders,disintegrating agents and the like. Because of their ease inadministration, tablets and capsules represent the most advantageousoral dosage form.

It is especially advantageous to formulate the compositions in unitdosage form for ease of administration and uniformity of dosage.Compositions in unit dosage form constitutes as aspect of the presentinvention. The term "unit dosage form" as used in the specification andclaims refer to physically discrete units, each unit containing apredetermined quantity of active ingredient calculated to produce thedesired therapeutic effect in association with the pharmaceuticalcarrier. Examples of such unit dosage forms are tablets, capsules,pills, powder packets, wafers, measured units in ampoules or inmultidose containers and the like. A unit dosage of the presentinvention will generally contain from 100 to 400 milligrams of thenon-steroidal antifungal agent and from about 50 to 400 milligrams ofthe azasterol compound, preferably about 100 to 200 milligrams of thenonsteroidal antifungal agent and 100 to 200 milligrams of the azasterolcompound.

The following examples illustrate novel compositions but are not to beconstrued as limiting:

EXAMPLE A

1000 compressed tablets each containing 100 milligrams ofN,N-dimethyl-3β-acetoxy 5,22(3)-choladiene-24-amine (Compound Ic) and200 milligrams of lovastatin are prepared from the followingformulation:

    ______________________________________                                                            Grams                                                     ______________________________________                                        Compound Ic           100                                                     Lovastatin            200                                                     Starch                750                                                     Dibasic calcium phosphate hydrous                                                                   5000                                                    Calcium stearate      2.5                                                     ______________________________________                                    

The finely powdered ingredients are mixed well and granulated with 10percent starch paste. The granulation is dried and compressed intotablets.

EXAMPLE B

1000 hard gelatin capsules, each containing 100 milligrams of25-azacholesterol and 200 milligrams of terbinafine are prepared fromthe following formulation:

    ______________________________________                                                       Grams                                                          ______________________________________                                        25-Azacholesterol                                                                              150                                                          Terbinafine      200                                                          Starch           250                                                          Lactose          750                                                          Talc             250                                                          Calcium stearate  10                                                          ______________________________________                                    

A uniform mixture of the ingredients is prepared by blending and used tofill two-piece hard gelatin capsules.

EXAMPLE C

1000 hard gelatin capsules, each containing 210 milligrams ofN,N-dimethyl-3β-hydroxy-5,22(Z)-choladiene-24-amine (Compound Ib) and290 milligrams of lovastatin are made by blending the followingcomposition and used to fill two-piece hard gelatin capsules.

    ______________________________________                                                       Grams                                                          ______________________________________                                        Compound Ib      210                                                          Lovastatin       290                                                          Starch           250                                                          Lactose          750                                                          Talc             250                                                          Calcium Stearate  10                                                          ______________________________________                                    

EXAMPLE D

In a similar manner 1000 gelatin capsules each containing 210 milligramsof 25-aza-22-oxacholesterol (Compound Id) and 290 milligrams of1-methyl-2-nonyl-5-(phenylmethyl)-3-pyrrolidinol (Compound C).

    ______________________________________                                                       Grams                                                          ______________________________________                                        Compound Id      210                                                          Compound C       290                                                          Starch           250                                                          Lactose          750                                                          Talc             250                                                          Calcium stearate  10                                                          ______________________________________                                    

EXAMPLE E

1000 compressed tablets each containing 200 milligrams of25-azacholesterol and 300 milligrams of5-(1-hydroxy-2,4,6-heptatriynyl)-2-oxo-1,3-dioxalane-4-heptanoic acidcarbonate (Compound B) are prepared in a manner similar to thatdescribed in Example A from the following formulation:

    ______________________________________                                                            Grams                                                     ______________________________________                                        25-Azacholesterol     200                                                     Compound B            300                                                     Starch                750                                                     Dibasic calcium phosphate hydrous                                                                   5000                                                    Calcium stearate      2.5                                                     ______________________________________                                    

EXAMPLE F

1000 hard gelatin capsules, each containing 210 milligrams of25-azacholesterol and 290 milligrams of11-(3-(hydroxymethyl)-4-oxo-2-oxetanyl)-7-methyl-2,4-undecadienoic acid(Compound A) are prepared in a manner similar to that described inExample B from the following formulation:

    ______________________________________                                                       Grams                                                          ______________________________________                                        25-Azacholesterol                                                                              200                                                          Compound A       290                                                          Starch           250                                                          Lactose          750                                                          Talc             250                                                          Calcium stearate  10                                                          ______________________________________                                    

What is claimed is:
 1. A fungicidal composition comprising(1) a25-azasterol compound having the formula ##STR19## wherein R is hydrogenor lower acyl, X is CH, CH₂ or O; the on the side chain indicates thatthe bond may be a single or a double bond provided that when X is O, orCH₂, it is a single bond; and (2) a HMG-CoA synthase inhibitor compoundin admixture with a pharmaceutically acceptable carrier.
 2. Acomposition according to claim 1 in unit dosage form in which the25-azasterol compound is present in an amount of from 50 to 200milligrams and the HMG-CoA synthase inhibitor compound is present in anamount of from 100 to 400 milligrams.
 3. A method for killing fungicausing mycotic infections comprising administering to the site infectedwith fungi, a composition of claim
 2. 4. A composition according toclaim 1 wherein the 25-azasterol compound is17β-[[3-(dimethylamino)propyl]methylamino]androst-5-en-3β-ol(25-azacholesterol) and the HMG-CoA synthase inhibitor compound is11-(3-(hydroxymethyl)-4-oxo-2-oxetanyl)-7-methyl-2,4-undecadienoic acid.5. A method for treating subjects with mycotic infections to eradicatethe disease causing fungi comprising administering to such subject afungicidally effective amount of a composition of claim 4.